Endogenous metabolite

N-(1,3-Dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) and its quinone derivative, 6PPD-quinone (6PPD-Q), have been found to be prevalent in the environment, but there are currently no data on their presence in humans. Herein, we conducted the first human biomonitoring study of 6PPD and 6PPD-Q by measuring 150 urine samples collected from three different populations (general adults, children, and pregnant women) in South China. Both 6PPD and 6PPD-Q were detected in the urine samples, with detection frequencies between 60% and 100%. Urinary 6PPD-Q concentrations were significantly higher than those of 6PPD and correlated well with those of 6PPD (p < 0.01), indicating coexposure to 6PPD and 6PPD-Q in humans. In vitro metabolic experiments demonstrated rapid depletion of 6PPD by human liver microsomes, which should be responsible for the lower concentrations of 6PPD in human urine. Additionally, pregnant women exhibited apparently higher concentrations of 6PPD and 6PPD-Q (median 0.068 and 2.91 ng/mL, respectively) than did adults (0.018 and 0.40 ng/mL) and children (0.015 and 0.076 ng/mL). The high daily urinary excretion of 6PPD-Q in pregnant women was estimated to be 273 (ng/kg bw)/day. Considering that 6PPD-Q was a lethal toxicant to multiple aquatic species, the potential human health risks posed by its long-term exposure require urgent attention[1].

Daily Excretion of 6PPD and 6PPD-Q in Urine [1]
Based on the urinary analyte concentrations measured in the three populations, the daily excretion of 6PPD and 6PPD-Q in urine was estimated using a model as described elsewhere, (49−51) i.e., daily excretion of 6PPD and 6PPD-Q in urine ((ng/kg bw)/day) = urinary analyte concentration (ng/mL) × daily excretion volume of urine (mL/day)/body weight (kg). In our estimation, values for daily excretion volume of urine were adopted from refs (50and51): 1700 mL/day for adults, 660 mL/day for children, and 2000 mL/day for pregnant women. Values for body weight were based on the average body weight obtained in our questionnaires: 60 kg for adults, 23 kg for children, and 64 kg for pregnant women (Table S2). Median and 95th concentrations of 6PPD and 6PPD-Q were used to calculate the median and high daily excretion, respectively. As shown in Figure 2, as expected, the daily excretion of 6PPD-Q in urine of adults, children, and pregnant women (median 11.3, 2.18, 90.9 (ng/kg bw)/day, respectively) was evidently higher than its parent 6PPD (median 0.51, 0.43, and 2.13 (ng/kg bw)/day, respectively). In addition, pregnant women had higher daily excretion of 6PPD and 6PPD-Q than did adults and children, and the high daily excretion of 6PPD-Q in urine of pregnant women was up to 247 (ng/kg bw)/day.
It should be pointed out that due to the difficulties in collecting 24-h urine samples, the estimates of daily excretion were limited by the reliance on spot morning urine samples, as there may be daily variance in urinary 6PPD and 6PPD-Q levels. Therefore, although many previous studies suggest that early morning urine is a feasible alternative to 24-h collections for estimating personal exposure, these results should be considered as a preliminary estimation. Despite the limitation, the daily urinary excretion of 6PPD and 6PPD-Q in these populations should be of concern because it largely reflects their internal exposure dose. Given that a portion of 6PPD and 6PPD-Q may also be excreted through feces and exhaled air like some other pollutants, the actual exposure dose of 6PPD and 6PPD-Q may even be higher than our estimates. Although the dose is unlikely to exceed a proposed reference dose (RfD) of 26000 (ng/kg bw)/day calculated for 6PPD, (56) the potential health risk posed by long-term exposure to 6PPD-Q requires considerable attention, as 6PPD-Q has been proven to be more toxic to aquatic organisms than 6PPD from the individual species to the tissue levels. In future studies, additional biomonitoring investigations on 6PPD and 6PPD-Q are needed to better elucidate their internal exposure in humans. More toxicological and epidemiological studies of 6PPD and 6PPD-Q are also recommended to shed light on their potential health risks.

[1]. First report on the occurrence of N-(1, 3-dimethylbutyl)-N’-phenyl-p-phenylenediamine (6PPD) and 6PPD-quinone as pervasive pollutants in human urine from south China. Environ Sci Technol Lett, 2022, 9(12): 1056-1062.

p-Phenylenediamine (PPD) compounds are an important class of synthetic antioxidants that have been massively used as additives for the manufacture of various rubber products, such as tires, footwear, and even food contact materials. Among the PPD compounds, N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) is one of the most frequently used species, which has been listed as a high production volume (HPV) chemical. In 2001, the global production of 6PPD was estimated to be 130000 tons. In 2020, its annual production in China alone reached as high as 200000 tons, which accounted for nearly 54% of the production of all rubbery antioxidants. With such a high yield, unfortunately, a recent study by Tian et al. suggested that 6PPD may be not safe. They discovered that 6PPD can be oxidized in the environment to form a toxic quinone derivative, called 6PPD-quinone (6PPD-Q), which was responsible for acute mortality of coho salmon (Oncorhynchus kisutch) in the Pacific Northwest. (6) Because of the ubiquitous use of 6PPD, this discovery has now triggered widespread scientific concern over the environmental contamination and toxic effects of 6PPD and its quinone derivative, 6PPD-Q.[1]
More recently, 6PPD and 6PPD-Q have been found in a variety of environmental matrices, including atmospheric particles, indoor dust, (5,11,12) road dust, playground dust, roadside soil, runoff water, and surface water. (16−18) In most cases, 6PPD and 6PPD-Q coexisted in these environmental matrices at comparable concentrations. For instance, Zhang et al. demonstrated ubiquitous distributions of 6PPD and 6PPD-Q in fine particulate matters (PM2.5) from six Chinese cities, and the atmospheric concentrations of 6PPD (median 0.9–8.4 pg/m3) were found to be similar to those of 6PPD-Q (median 1.7–6.7 pg/m3). Hiki et al. reported the coexistence of 6PPD and 6PPD-Q in road dust from Tokyo, Japan, and comparable concentrations of 6PPD (45–1175 ng/g) and 6PPD-Q (116–1238 ng/g) were also observed in the dust samples.[1]
Aside from environmental ubiquity, emerging evidence also suggested some significant toxicities of 6PPD and 6PPD-Q. (6,19−24) For example, 6PPD has been shown to induce toxicity in fathead minnow (Pimephales promelas) and freshwater mussel (Lampsilis siliquoidea). In addition to being highly toxic to coho salmon as reported by Tian et al., (6,21) 6PPD-Q was also demonstrated to be toxic to zebrafish larvae with a 24 h LC50 of 308.67 μg/L. More recently, 6PPD and 6PPD-Q were found to cause anxiety-like behaviors and unbalance in zebrafish. Although the aquatic toxicities of 6PPD and 6PPD-Q are becoming clearer, their adverse effects on mammals and humans remain largely unknown. In particular, internal exposure to 6PPD and 6PPD-Q may pose potential health risks to humans. However, despite the newfound ubiquity of 6PPD and 6PPD-Q in the environment, their occurrences, concentrations, and exposure status in humans are still unclear.[1]
To fill the knowledge gap, we conducted the first human biomonitoring study of 6PPD and 6PPD-Q by measuring urine samples, considering that urine plays an important role in the excretion of environmental pollutants and is also relatively easy to collect, store, and prepare. A total of 150 human urine samples were collected from three different populations (general adults, children, and pregnant women) in South China and analyzed for 6PPD and 6PPD-Q. The main objectives were to (1) determine the occurrences of 6PPD and 6PPD-Q in human urine, (2) compare the internal levels of 6PPD and 6PPD-Q in different populations from South China, and (3) provide baseline information on internal exposure of 6PPD and 6PPD-Q in humans. The results of this study will greatly improve our understanding of human exposure to 6PPD and 6PPD-Q.[1]

C18H22N2O2

298.38

298.168

2754428-18-5

154926030

Pink to red solid powder

4.1

2

4

6

22

485

0

C1(NC(CC(C)C)C)C(C=C(C(C=1)=O)NC1C=CC=CC=1)=O

UBMGKRIXKUIXFQ-UHFFFAOYSA-N

InChI=1S/C18H22N2O2/c1-12(2)9-13(3)19-15-10-18(22)16(11-17(15)21)20-14-7-5-4-6-8-14/h4-8,10-13,19-20H,9H2,1-3H3

2-anilino-5-(4-methylpentan-2-ylamino)cyclohexa-2,5-diene-1,4-dione

6PPD-quinone; 2754428-18-5; 6PPD-Q; 2-((4-Methylpentan-2-yl)amino)-5-(phenylamino)cyclohexa-2,5-diene-1,4-dione; 2,5-Cyclohexadiene-1,4-dione, 2-[(1,3-dimethylbutyl)amino]-5-(phenylamino)-; 6PPD quinone; 6PPD-Quinone; 2-((4-Methylpentan-2-yl)amino)-5-(phenylamino)cyclohexa-2,5-diene-1,4-dione; 6PPD-quinone; 6PPD-Quinone; 2-[(1,3-Dimethylbutyl)amino]-5-(phenylamino)-2,5-cyclohexadiene-1,4-dione (ACI); 2,5-Cyclohexadiene-1,4-dione, 2-[(1,3-dimethylbutyl)amino]-5-(phenylamino)- (ACI); G8MFB8G7B6;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)